(1) Field of the Invention
The present invention relates to an absorbent composed of porous beads of chitosan. More particularly, the present invention relates to an adsorbent composed of porous beads of uncrosslinked or crosslinked chitosan to which protein A or lectin is bonded through a combination group having a carboxyl group.
Furthermore, the present invention relates to a method for removing immunoglobulin by using an adsorbent composed of porous beads of chitosan, and to a method in which adsorption by affinity chromatography or removal of an inhibitor for the activity of interleukin-2 is accomplished by an adsorbent composed of porous beads of uncrosslinked or crosslinked chitosan to which a carboxyl group is bonded, or an adsorbent composed of porous beads of uncrosslinked or crosslinked chitosan to which protein A or lectin is bonded through an amide group.
(2) Description of the Related Art
In affinity chromatography, a chromatographical technique, separation or purification is performed by utilizing the affinity between a pair of substances specifically exerting mutual actions on each other. For example, affinity chromatography is valuable for purifying a biological substance on the basis of a discrimination of a biological characteristic of the biological substance, that is, a specific chemical structure on the molecule.
An adsorbent (i.e., affinity gel) for the affinity chromatography comprises, for example, an active support obtained by bonding a combination group (spacer) to an insoluble carrier (matrix) and a ligand bonded to the spacer. The adsorption operation is performed on a combination of this ligand and a selected substance, which exert mutual actions on each other.
As the combination of the ligand and the intended substance to be adsorbed, there can be mentioned a combination of an enzyme and a substrate, product, inhibitor, coenzyme or effector, a combination of an antigen and an antibody, a combination of a receptor and an agonist, a pair of a nucleic acid and a base, a combination of lectin and a saccharide or glycoprotein, a combination of a metal chelate and a protein, a combination of a hydrophobic group and a protein, a combination of a host and a guest, and a combination of protein A and immunoglobulin G (IgG).
In the separation, purification or analysis by affinity chromatography, the active support, which is the main constituent of the adsorbent for the affinity chromatography, must have the following properties. Namely, a small non-specific adsorption, a high porosity, an easily accomplished bonding of the ligand, a large fixation-capacity, a high chemical stability such that the support is stable and a change of the volume does not occur within broad ranges of the pH value, salt concentration and temperature, a support having a required mechanical strength and stability, good flowability characteristics, and a high resistance to biological contamination.
Cellulose, dextran, polyacrylamide and agarose customarily used as the substrate of the adsorbent for the affinity chromatography do not possess all of these required properties. In particular, since they are soft gels having a poor hardness, their flowability characteristics and separation characteristics are not satisfactory. Moreover, they have a very short life.
The silica beads recently used have a satisfactory hardness, but since they cannot be used under alkaline conditions, selection of the separation conditions or the eluting and washing conditions is considerably restricted.
It is known that protein A adsorbs IgG or an immune complex (Immunochemistry, vol. 7, pages 124 through 127), and it is also known that an immune complex acts as a blocking factor in the immune system. Accordingly, it is presumed that the adsorption of the immune complex by an adsorbent bearing protein A is related to the activation of the immune system, but the action of protein A on an interleukin-2 (IL-2) inhibitor and immunosuppresive substances has not been fully elucidated. As one specific and practical medical application of protein A, a fixation of protein A to an appropriate carrier has been attempted: For example, there have been proposed an adsorbent formed by coating protein A on the surface of active carbon, an adsorbent formed by embedding protein A in a collodion membrane (Engl. J. Med., 305, 1195, 1981), and an adsorbent formed by fixing protein A to a polysaccharide such as agarose or dextran or a plastic material such as polystyrene.
However, these adsorbents have the following problems.
(1) The amount of protein A fixed is small and the adsorption capacity is low.
(2) The fixation of the protein A is not stable, and there is a risk of a leakage of the protein A or the production of a harmful side effect on a human body.
(3) Since the carrier has a poor affinity with a living body, coagulation is often caused by contact with a body fluid such as blood.
(4) If a carrier has a high hydrophobic property, such as a polystyrene carrier, albumin, globulin and the like are non-specifically adsorbed and the capacity of selective adsorption by protein A is reduced.
When protein A is fixed by ionic adsorption, physical adsorption or embedding, leakage of the protein A occurs and it is possible that this will prove harmful to the human body. Accordingly, the development of a method for fixing protein A quantitatively and stably on a carrier is required. On the other hand, if the supporting carrier is recognized as a foreign substance in a living body, blood is coagulated or an antibody is induced around the carrier. Accordingly, the contact with blood is inhibited and the effect is reduced, and clots of coagulated blood migrate in the blood vessel and produce thrombus in a narrow portion thereof. Therefore, it is possible that this could have a serious effect dangerous to a living body.